Molecular typing of clinical multidrug-resistant Klebsiella pneumoniae isolates.

INTRODUCTION: Klebsiella pneumoniae is an opportunistic pathogen which is an important cause of hospital-acquired and antibiotic resistance infections. Therefore, this study aimed to determine the frequency of resistance to antibiotics, as well as the molecular typing of the associated isolates, and compare multiple-locus VNTR analysis (MLVA) and Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) methods to specify the degree to which distinctions can be separated from each other. METHODS AND MATERIALS: One hundred K. pneumoniae isolates were obtained from different sources of infections from patients admitted to hospitals. Antibiotic susceptibility testing was then performed by applying the Kirby-Bauer disk diffusion method. Typing of K. pneumoniae was done by utilizing MLVA and ERIC-PCR methods. RESULTS: Eighty-six multidrug-resistant (MDR) K. pneumoniae isolates were identified, which resistance to ampicillin, trimethoprim/sulfamethoxazole, and ceftriaxone was the most frequent in the considered isolates (100, 93, and 93%, respectively). A total of 50 different antibiotic susceptibility patterns were observed among the MDR K. pneumonia, with the most frequent pattern being resistance to all antibiotics (12.79%) and resistance to all antibiotics except amikacin (10.47%). The isolates were then divided into 37 different MLVA types and seven clonal complexes were obtained from the minimum spanning tree analysis. Finally, the isolates were assigned to 38 different ERIC types. The discriminatory power of MLVA and ERIC methods also showed a value of 0.958, and 0.974. CONCLUSION: Both PCR-typing methods with phenotypic patterns can be useful for the epidemiological typing of K. pneumoniae isolates with the highest performance in discriminating isolates.

Peptide nucleic acid-mediated re-sensitization of colistin resistance Escherichia coli KP81 harboring mcr-1 plasmid.

Escherichia coli is a gram-negative bacterium and it causes a variety of diseases in humans. It causes a wide range of clinical infections in humans; urinary tract infections is the most prevalent infection caused by uropathogenic Escherichia coli. In recent years, the observation of antibiotic-resistant genes such as resistance to colistin, makes the Escherichia coli resistant to antibiotics like colistin (polymyxin E), because of that the use of new therapies like peptide nucleic acid (PNA) has attracted the consideration of scientists. The aim of this study is the assessment of the inhibitory role of PNA against mcr-1 gene and reduction of mcr-1 gene expression and MIC in colistin resistant E. coli by PNA. NCBI database was used to design PNA. Our study was carried out on E. coli KP81 bacteria containing the mcr-1 gene. Microbroth dilution (MIC) method was used to survey phenotypic sensitivity and determine the sensitivity of the bacteria to the colistin antibiotic. E. coli KP81 isolates were further investigated by polymerase chain reaction to assess the presence of mcr-1 genes and target genes were quantified by real-time PCR assay using specific primers. The MIC result after treatment with specific PNA showed that the resistance to colistin reduced about three fold and the resistance level dropped from 32 µg/ml to 4 µg/ml. The expression analysis of mcr-1 gene in E. coli KP81 isolate indicates the PNA, 95% reduced the expression of the mcr-1 gene. Our observations showed that by inhibiting the expression of mcr-1, sensitivity to colistin can be defeated. Using higher concentrations of PNA and an in vivo study can reveal more clinical application of this method.

Addition of Bioactive Glass Decreases Setting Time and Improves Antibacterial Properties of Mineral Trioxide Aggregate.

Objectives: This study aimed to assess the effect of addition of bioactive glass (BG) on the setting time and antibacterial activity of mineral trioxide aggregate (MTA) against Enterococcus faecalis (E. faecalis). Materials and Methods: In this in vitro study, BG was synthesized by the sol-gel technique and added to MTA powder in certain ratios. Three groups of specimens were fabricated from pure MTA, MTA mixed with 10wt% BG, and MTA mixed with 20wt% BG. The setting time of specimens was measured according to ISO9917-2007. Direct contact test was used to assess the antimicrobial activity of the three groups against E. faecalis. Data were analyzed by repeated measures ANOVA (alpha = 0.05). Results: Addition of BG (in both concentrations) to MTA decreased its setting time and improved its antibacterial activity against E. faecalis (p < 0.05). By an increase in concentration of BG (20%), the antimicrobial activity further improved (p < 0.05). Conclusion: Addition of BG to MTA in 10wt% and 20wt% concentrations decreased its setting time and improved its antibacterial activity against E. faecalis.

MALDI-TOF Mass Spectroscopy Applications in Clinical Microbiology.

There is a range of proteomics methods to spot and analyze bacterial protein contents such as liquid chromatography-mass spectrometry (LC-MS), two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), which give comprehensive information about the microorganisms that may be helpful within the diagnosis and coverings of infections. Microorganism identification by mass spectrometry is predicted on identifying a characteristic spectrum of every species so matched with an outsized database within the instrument. MALDI-TOF MS is one of the diagnostic methods, which is a straightforward, quick, and precise technique, and is employed in microbial diagnostic laboratories these days and may replace other diagnostic methods. This method identifies various microorganisms such as bacteria, fungi, parasites, and viruses, which supply comprehensive information. One of the MALDI-TOF MS's crucial applications is bacteriology, which helps identify bacterial species, identify toxins, and study bacterial antibiotic resistance. By knowing these cases, we will act more effectively against bacterial infections.

Evaluation of Resistance Mechanisms in Carbapenem-Resistant Enterobacteriaceae.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) is a major concern leading to morbidity and mortality in the world. CRE often is becoming a cause of therapeutic failure in both hospital and community-acquired infections. AIM: This study aimed to investigate the resistance mechanisms of CRE by phenotypic and molecular methods. MATERIALS AND METHODS: Sixty CRE (50 Klebsiella pneumoniae, 6 Escherichia coli, and 4 Enterobacter spp.) were isolated from October 2018 to June 2019. Antimicrobial susceptibility testing was carried out using phenotypic methods. The carbapenem resistance mechanisms including efflux pump hyperexpression, AmpC overproduction, carbapenemase genes, and deficiency in OmpK35 and OmpK36 were determined by phenotypic and molecular methods, respectively. RESULTS: Sixty CRE (50 Klebsiella pneumoniae, 6 Escherichia coli, and 4 Enterobacter spp.) were isolated from October 2018 to June 2019. Amikacin was found to be the most effective drug against CRE isolates. All isolates were resistant to imipenem and meropenem by the micro-broth dilution. AmpC overproduction was observed in all Enterobacter spp. and three K. pneumoniae isolates. No efflux pump activity was found. Carba NP test and Modified Hodge Test could find carbapenemase in 59 (98%) isolates and 57 (95%) isolates, respectively. The most common carbapenemase gene was bla OXA-48-like (72.8%) followed by bla NDM (50.8%), bla IMP (18.6%), bla VIM (11.8%), and bla KPC (6.7%). The ompK35 and ompK36 genes were not detected in 10 and 7 K. pneumoniae isolates, respectively. CONCLUSION: The amikacin is considered as a very efficient antibiotic for the treatment of CRE isolates in our region. Carbapenemase production and overproduction of AmpC are the main carbapenem resistance mechanisms in CRE isolates. Finally, Carba NP test is a rapid and reliable test for early detection of carbapenemase-producing isolates.

Frequency of DNA gyrase and topoisomerase IV mutations and plasmid-mediated quinolone resistance genes among Escherichia coli and Klebsiella pneumoniae isolated from urinary tract infections in Azerbaijan, Iran.

OBJECTIVES: This study assessed genetic alterations in gyrA, gyrB, parC and parE and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes among Escherichia coli and Klebsiella pneumoniae isolates from urinary tract infections (UTIs) in Azerbaijan, Iran. METHODS: A total of 205 clinical isolates of E. coli (n=177) and K. pneumoniae (n=28) were obtained from UTIs. Antimicrobial susceptibility was determined by disk diffusion and agar dilution assays. The presence of PMQR genes was determined by PCR, and sequencing of the gyrA, gyrB, parC and parE was performed. RESULTS: The rate of fluoroquinolone (FQ) resistance among the isolates was 77.1%. The Ser83Leu mutation in gyrA was observed in all 60 FQ-resistant isolates selected for direct sequencing. The second most common mutation in gyrA was Asp87Asn. Frequent mutations in parC were Ser80Ile and Glu84Val. Ser359Ala+Ser367Thr and Gly385Cys mutations in gyrB were identified in one isolate each of K. pneumoniae and E. coli, respectively. The parE gene had mutations at Ile529Leu, Ser458Ala and Leu416Phe. Overall, PMQR determinants were identified in 90% of E. coli and 100% of K. pneumoniae. The prevalence of PMQR genes was as follows: aac(6')-Ib-cr, 71.7%; oqxB, 51.7%; oqxA, 36.7%; qnrB, 28.3%; qnrS, 21.7%; qnrD, 16.7%; qepA, 5.0%; qnrA, 1.7%; and qnrC, 1.7%. CONCLUSIONS: FQ resistance rates were high. Mutations in DNA gyrase and topoisomerase IV and the prevalence of PMQR genes in E. coli and K. pneumoniae isolates were alarming. Moreover, the combination of these resistance mechanisms plays an important role in high-level FQ resistance.

The prevalence of plasmid-mediated quinolone resistance and ESBL-production in Enterobacteriaceae isolated from urinary tract infections.

INTRODUCTION: ß-lactam and fluoroquinolone antibiotics are usually used for the treatment of urinary tract infections (UTIs). The aim of this study was to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) and extended spectrum ß-lactamases (ESBLs) in Enterobacteriaceae isolated from UTIs. MATERIALS AND METHODS: Two hundred and nineteen samples of Enterobacteriaceae isolated from UTIs were collected in the Northwest of Iran. Antimicrobial susceptibility testing was determined by the disk diffusion method. ESBLs were detected by the double-disk test. ESBL and PMQR-encoding genes were screened using the polymerase chain reaction. RESULTS: The rate of resistance to moxifloxacin, nalidixic acid, gatifloxacin, ofloxacin, ciprofloxacin, and levofloxacin in ESBL-producing isolates was 89.3%, 88%, 84%, 80%, 78.7%, and 73.3%, respectively. PMQR-producing Enterobacteriaceae isolates were identified in 67 samples (89.1%). The most prevalent PMQR genes were aac (6')-Ib-cr 120 (68.6%) followed by oqxB 72 (41.1%), oqxA 59 (33.7%), qnrB 36 (20.6%), qnrS 33 (18.9%), qnrD 19 (10.9%), qepA 13 (7.4 %), qnrA 10 (5.7%), and qnrC 9 (5.1%). There was a strong association between PMQR genes and blaCTX-M-15 and blaTEM-116 and other ESBL genes. CONCLUSION: High resistance rates were detected to quinolones among ESBL-producing isolates from UTIs. There is a high prevalence of PMQR genes in Enterobacteriaceae in Azerbaijan and Iran, and the most common PMQR is aac(6')-Ib-cr. There is a significant association between PMQR and ESBL-producing isolates.

Prevalence of integrons 1, 2, 3 associated with antibiotic resistance in Pseudomonas aeruginosa isolates from Northwest of Iran.

BACKGROUND: The presence of Class 1, 2 and 3 integrons in clinical isolates of Pseudomonas aeruginosa with multi-drug resistance phenotype has rendered the organism as a new concern. OBJECTIVE: This study aimed to investigate the prevalence of Class 1, 2 and 3 integrons in multi-drug resistant clinical isolates of Pseudomonas aeruginosa collected from hospitals in the city of Tabriz Materials and Methods: A total of 200 P. aeruginosa non-duplicated clinical isolates were collected from inpatients and outpatients in different wards of hospitals from May to November 2016. The bacteria were identified by conventional microbiological methods. Antibiotic susceptibility test was performed by disk diffusion method and the presence of integrons was analyzed by polymerase chain reaction (PCR). RESULTS: Colistin was the most effective antibiotic, while 98% of the isolates were resistant to cefotaxime. Fifty-three percent of the isolates were recorded as multi-drug resistant (MDR) phenotype; however, 27.5% of the isolates were resistant to more than 8 antibiotics. In this study, 55 (27.5%), 51 (25.5%), and 30 (15%) clinical isolates of P. aeruginosa were positive for Class 1, 2 and 3 integrons, respectively. aac(6)II in Class I integrons and dfrA1 in ClassII and aacA7 in Class II integrons were the most prevalent genes. Resistance to aminoglycosides were the most common genes harbored by integrons. CONCLUSION: The results of this study showed that the prevalence of Class 1, 2 and 3 in integron genes in most P. aeruginosa strains islated from different parts and equipment used in the hospital. The role of these transferable genetic agents has been proven in the creation of resistance. Therefore, it is essential to use management practices to optimize the use of antibiotics, preferably based on the results of antibiogram and trace coding genes for antibiotic resistance.

Evaluation of Antimicrobial Effects of Different Concentrations of Triple Antibiotic Paste on Mature Biofilm of Enterococcus faecalis.

Background and aims. Triple antibiotic paste (TAP) is widely used in endodontics for root canal disinfection, particularly in regenerative procedures. The aim of this in vitro study was to evaluate the antimicrobial effects of different concentrations of TAP at 1-, 2-, 3-, and 4-week intervals on mature Enterococcus faecalis biofilm. Materials and methods. A total of 287 extracted one-rooted human central incisors were infected with E. faecalis ATCC 29212 after removing the crown and preparation. The root canal space was filled with one of the 0.01-, 0.1-, 1-, 10-, 100-, and 1000-mg/mL concentrations of TAP or normal saline (control). The root canal dentin was sampled after 1, 2, 3, and 4 weeks. The dentinal shavings were cultured on Mueller-Hinton agar plates after serial dilutions. The classic colony-forming unit (CFU) counting technique was used to determine remaining bacterial counts. Data were analyzed by using the two-way ANOVA, post hoc Tukey tests and one-way ANOVA (P<0.05). Results. TAP completely eliminated E. faecalis biofilms at all the intervals at concentrations of 1000, 100, and 10 mg/mL, whereas 1-, 0.1-, and 0.01-mg/mL TAP resulted in significant reduction of CFU means compared with the control group. There were no statistically significant differences between the four time intervals. Conclusion. Use of lower concentrations of TAP at short term could eradicate E. faecalis biofilm and decrease high-concentration side effects.

Bactericidal effects of Nd:YAG laser irradiation and sodium hypochlorite solution on Enterococcus faecalis biofilm.

OBJECTIVE: The aim of this study was to evaluate the bactericidal effects of Nd:YAG laser on biofilm of Enterococcus faecalis. BACKGROUND DATA: It is difficult to eliminate bacterial biofilms with routine endodontic preparation techniques. It might be possible to eliminate biofilms remaining in the root canals of teeth with lasers. MATERIALS AND METHODS: The root canals of 60 extracted teeth were prepared and E. faecalis biofilms were formed within the root canals. Then the teeth were randomly divided into four groups of 15. Group 1 samples did not undergo any interventions, to serve as controls. Group 2 samples underwent a 3-W laser beam for 10 sec. The root canals in group 3 were irrigated with 1% sodium hypochlorite for 15 min and then irradiated with a 3-W laser beam for 10 sec. The root canals in group 4 were irrigated with 1% sodium hypochlorite for 15 min. Dentin chips were collected from the root canal walls and weighed. Then the chips were used to prepare a suspension. The classic colony-forming unit (CFU) counting technique was used to determine remaining bacterial counts. RESULTS: The bacterial counts in groups 2 and 4 had decreased to 54% and 2.39% of the control group, respectively. In group 3 no bacterial growth was observed. There were no significant differences between groups 1 and 2 (p>0.05). CONCLUSIONS: Based on the results of the present study, the effect of Nd:YAG laser beam on E. faecalis biofilm is less than that of sodium hypochlorite solution. A combination of laser and sodium hypochlorite results in complete elimination of E. faecalis biofilm.